ABSTRACT Tens of thousands of people die each year from opioid overdose. Many of these people began taking opioids for pain. A critical treatment goal is to reduce the development of opioid dependence either by enhancing opioid analgesia so lower doses can be used or by blocking withdrawal symptoms. Opioid substitution therapy, in which long-lasting opioids such as methadone and buprenorphine are substituted for potent short acting opioids, requires continuous administration to mask opioid withdrawal without reducing opioid dependence. A potentially new approach is suggested by the finding that chronic opioid administration increases the formation of the mu-delta opioid receptor heterodimer (MDOR), and disrupting signaling from these heterodimers appears to enhance opioid antinociception and reduce dependence. These findings suggest that an MDOR antagonist may be especially effective in reducing dependence by limiting opioid tolerance and preventing opioid withdrawal. We created a novel series of potential selective peptide MDOR antagonists to test this hypothesis. These novel antagonists connect low affinity MOR (H-Tyr-Pro-Phe-D1Nal-NH2) and moderate affinity DOR (Tyr-Tic-OH) pharmacophores with a variable length (15-42 atom) flexible polyamide spacer. Our preliminary in vitro data using radioligand binding and 35S-GTP?S shows selectively targeting of the MDOR, with a selectivity ratio of ~89 fold for our best compound, D24M. Microinjection of D24M into the mouse brain selectively increased opioid antinociception in models of acute and chronic pain while strongly decreasing morphine withdrawal. These preliminary findings suggest that D24M could reduce opioid dependence by enhancing opioid antinociception, reducing opioid tolerance, or directly inhibiting opioid withdrawal. Although this completely new class of ligand is promising, the efficacy and translatability of MDOR antagonists depends on the ability to reduce dependence in the absence of disruptive side effects. The UG3 phase of this application will vigorously assess the ability of D24M to reduce dependence in male and female mice and rats. Indications supported by mouse models will be tested using the highly novel home cage wheel-running test in rats to determine the effect of D24M on normal daily function. Male and female rats with and without chronic pain will be included to mimic the clinical situation of pain patients who transition to dependence. If these studies are successful in showing that the D24M can reduce dependence (the UG3 milestone), then new derivatives of D24M to improve MDOR potency, selectivity, metabolic stability, and blood-brain barrier (BBB) penetration via glycosylation and nanoparticle formulation will be developed (UH3 phase). The ultimate goal is to develop an optimized drug ready for Investigational New Drug (IND)-enabling studies and clinical testing. These proposed studies are high-risk, high-reward, with a brand new class of therapeutic drugs to be tested in highly innovative rodent behavioral models. Our plan to assess D24M, the novel MDOR antagonist, and develop new drugs to reduce opioid dependence is well suited to the UG3/UH3 mechanism.